Objective: To optimize a workflow for extracting human deep brain RNA from DBS microelectrodes for transcriptome sequencing.

Background: Understanding the molecular mechanisms of Parkinson’s disease (PD) has largely relied on post-mortem studies, cerebrospinal fluid biomarkers, and animal models due to limited access to living human brain tissue. Deep brain stimulation (DBS) surgery offers a unique opportunity to obtain trace brain tissue from disposable recording microelectrodes, enabling minimally disruptive RNA collection for in-vivo human brain transcriptomic analysis.

Method: Protocol optimization used unfixed post-mortem human brains. Recording microelectrodes were inserted, withdrawn, and placed in RNA lysis buffer to recover tissue. RNA was extracted using a modified RNeasy Micro Kit protocol, assessed with a Bioanalyzer, and libraries were prepared using a low-input RNA library kit. Libraries were sequenced on the NovaSeq 6000, and RNA-Seq data were analyzed on the Galaxy platform using a GRCh38 reference-based workflow including QC, alignment, quantification, and DESeq2 analysis.

Results: Three post-mortem brains were used for protocol optimization, with 38 microelectrodes introduced. Twelve single MEs and thirteen pooled ME pairs yielded 25 RNA isolates. Among 11 isolates assessed, 54.5% showed RIN ≥5, with a median RIN of 6.4 (range 1–7.8). The mean RNA concentration was 145.6 ± 105.1 pg/µL. Of the 25 isolates, 20 were processed for library preparation, resulting in 18 successful cDNA libraries. Fifteen libraries representing 24 microelectrodes passed sequencing quality thresholds. Sequencing metrics demonstrated high data quality (mean 41.7M reads, Q30 = 83.2%, GC content 47.3%). Analysis of top 50 highly expressed genes confirmed the presence of several genes with high brain tissue expression (Figure.2). Principal component analysis showed clear clustering of transcriptomes corresponding to autopsy brain groups, indicating strong within-group similarity and inter-group differences (Figure. 3).

Conclusion: This study shows that RNA can be isolated from deep brain tissue on DBS microelectrodes using a low-input workflow. The RNA is suitable for transcriptomic analysis and enables in-vivo molecular profiling of the human brain without altering DBS surgery, opening new avenues to study mechanisms and biomarkers in Parkinson’s disease and other movement disorders.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/an-optimized-workflow-for-high-throughput-rna-seq-from-human-deep-brain-tissue-using-dbs-microelectrodes/